Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection.
A highly sensitive method for the determination of alpha-keto acids including phenylpyruvic acid in human plasma is investigated. The method employs high-performance liquid chromatography with chemiluminescence detection. The acids and alpha-ketocaproic acid (internal standard) in human plasma are isolated by anion-exchange chromatography on a Toyopak DEAE cartridge, and then converted into the corresponding chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 50 min on a reversed-phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III). The detection limits for the acids are in the range 9-92 pmol/ml in plasma (signal-to-noise ratio = 3). This sensitivity permits precise determination of several alpha-keto acids including phenylpyruvic acid, which cannot be determined by other HPLC methods, in 10 microliters of normal human plasma. The chemiluminescent product from phenylpyruvic acid was characterized as 3-benzyl-7,8-dihydropyridazino[4,5-g]quinoxaline-2,6,9(1H)-trione.
Nakahara
T
T
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Ishida
J
J
Yamaguchi
M
M
Nakamura
M
M
D000328:Adult; D002208:Caproates / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D007651:Keto Acids / Q000097:blood*; D008163:Luminescent Measurements; D008297:Male; D010667:Phenylpyruvic Acids / Q000097:blood*; D010793:Phthalazines; D010949:Plasma / Q000737:chemistry*; D015203:Reproducibility of Results
D002208:Caproates; D007651:Keto Acids; D010667:Phenylpyruvic Acids; D010793:Phthalazines; C001916:alpha-ketocaproic acid; C067545:4,5-diaminophthalhydrazide; C031606:phenylpyruvic acid